Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channels are the major apical exit pathways for anion (chloride and bicarbonate) secretion from the brush border membrane (BBM) in the intestine. Absence of CFTR on the enterocyte BBM leads to hyperacidity and intestinal obstruction as observed in the disease Cystic Fibrosis (CF). Increased CFTR on the BBM leads to secretory diarrhea. Our studies identified a unique subcellular distribution for CFTR in the intestine that is important fo both basal and increased fluid secretion under pathophysiologic conditions such as bacterial enterotoxins and low luminal pH. Unlike other epithelia that express CFTR only on the BBM, enterocytes express CFTR both on the BBM and subapical endosomes. The BBM fraction maintains basal intestinal fluidity by constitutive recycling of CFTR. The larger fraction of CFTR in subapical endosomes is important for rapid stress responses. For example, bacterial enterotoxins increase intracellular cyclic nucleotides that lead to rapid insertion of endosomal CFTR into the BBM to increase the number of CFTR channels and elicit massive fluid secretion. Similarly, luminal acidity elicits robust CFTR traffic into the BBM of villus enterocytes in the smll intestine to secrete bicarbonate. Thus, luminal pH in the CF small intestine is acidic. The protein motors and factors that direct CFTR traffic into and out of the enterocyte BBM are not known and what is known is largely credited to this laboratory. Previous investigations revealed that a minus motor Myosin6 (Myo6) directs CFTR endocytosis from the BBM of crypt and villus enterocytes. During the previous R01 cycle, the specific adaptor (AP-2-) that links CFTR to the Myo6 motor was identified. Proposed studies will investigate the site of CFTR- AP-2 binding, elucidate the mechanism and site of action of AP-2 in the intestine. Myo6 operates in tandem with its counterpart plus end motor Myosin 1a (Myo1a) in the enterocyte. Proposed studies in the first application sought to investigate the role of Myo1a in CFTR BBM localization and function in Myo1a KO mice. These studies were published. Myo1a is the exocytic motor for CFTR traffic from endosomes into the BBM of villus enterocytes. The mechanisms regulating Myo1a-dependent exocytosis of CFTR will now be examined. New studies in Aim 3 will use a Myo6/Myo1a double KO mouse model to test the hypothesis that Myo6 and Myo1a function as counterpart motors to maintain proper CFTR BBM localization and function by endocytic and exocytic traffic into and out of the enterocyte BBM. Proposed studies will expand critical knowledge gained during the previous cycle on factors and mechanisms regulating CFTR in the intestine under homeostatic and pathophysiologic conditions and extend insights into CFTR trafficking mechanisms that can be targeted to treat common intestinal diseases such as constipation.